Process for the preparation of a growth hormone secretagogue

ABSTRACT

The present invention is directed to a novel process for the preparation of the compound N- 1(R)- (1,2-dihydro-1-methanesulfonyl-spiro 3H-indole-3,4&#39;-piperdin!-1&#39;-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methyl-propanamide, and salts thereof, which has the structure: ##STR1## and which has the ability to stimulate the release of natural or endogenous growth hormone. This compound may be used to treat conditions which require the stimulation of growth hormone production or secretion such as in humans with a deficiency of natural growth hormone or in animals used for food or wool production where the stimulation of growth hormone will result in a larger, more productive animal.

This application is based on provisional application No. 60/005,898filed Oct. 27, 1995.

BACKGROUND OF THE INVENTION

Growth hormone, which is secreted from the pituitary, stimulates growthof all tissues of the body that are capable of growing. In addition,growth hormone is known to have the following basic effects on themetabolic processes of the body: (1) Increased rate of protein synthesisin all cells of the body; (2) Decreased rate of carbohydrate utilizationin cells of the body; (3) Increased mobilization of free fatty acids anduse of fatty acids for energy. A deficiency in growth hormone secretioncan result in various medical disorders, such as dwarfism.

Various ways are known to release growth hormone. For example, chemicalssuch as arginine, L-3,4-dihydroxyphenylalanine (L-DOPA), glucagon,vasopressin, and insulin induced hypoglycemia, as well as activitiessuch as sleep and exercise, indirectly cause growth hormone to bereleased from the pituitary by acting in some fashion on thehypothalamus perhaps either to decrease somatostatin secretion or toincrease the secretion of the known secretagogue growth hormonereleasing factor (GRF) or an unknown endogenous growth hormone-releasinghormone or all of these.

In cases where increased levels of growth hormone were desired, theproblem was generally solved by providing exogenous growth hormone or byadministering GRF or a peptidal compound which stimulated growth hormoneproduction and/or release. In either case the peptidyl nature of thecompound necessitated that it be administered by injection. Initiallythe source of growth hormone was the extraction of the pituitary glandsof cadavers. This resulted in a very expensive product and carried withit the risk that a disease associated with the source of the pituitarygland could be transmitted to the recipient of the growth hormone.Recombinant growth hormone has become available which, while no longercarrying any risk of disease transmission, is still a very expensiveproduct which must be given by injection or by a nasal spray. Othercompounds have been developed which stimulate the release of endogenousgrowth hormone.

In particular, certain spiro compounds are disclosed in PCT PatentPublication WO 94/13696 and Proc. Natl. Acad. Sci. USA, 92, 7001-7005(July 1995) as being non-peptidal growth hormone secretagogues. Thesecompounds have the ability to stimulate the release of natural orendogenous growth hormone and thus may be used to treat conditions whichrequire the stimulation of growth hormone production or secretion suchas in humans with a deficiency of natural growth hormone or in animalsused for food or wool production where the stimulation of growth hormonewill result in a larger, more productive animal.

Among the preferred compounds disclosed therein is spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamidewhich has the structure: ##STR2##

PCT Patent Publication WO 94/13696 discloses methods for preparing thiscompound (see Examples 18, 19 and 55). However, the synthesis of thecompound was accomplished by using the very expensive amino acidcoupling agent EDC ($1100/kg); the use of numerous equivalents oftrifluoroacetic acid as the catalyst for the BOC group deprotections;extensive chromatographic purifications; and resulted in "gumming" ofthe final product.

The advantages of the present invention include: a 6-step high yieldingnon-isolation process providing material of ≦99.9% purity; decreasedexpense through the use of DCC $40/kg! instead of EDC $1100/kg!;diminished environmental impact through the use of methanesulfonic acidinstead of trifluoroacetic acid as the catalyst (as well as lesserequivalents of catalyst) in the deprotections; and ease of isolation ofthe final product.

SUMMARY OF THE INVENTION

The instant invention is directed to a process for the preparation ofthe compound N- 1(R)- (1,2-dihydro-1-methanesulfonyl-spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methyl-propanamidewhich has the structure: ##STR3## and salts thereof, in particular, themethanesulfonate salt.

This compound has the ability to stimulate the release of natural orendogenous growth hormone and may be used to treat conditions whichrequire the stimulation of growth hormone production or secretion suchas in humans with a deficiency of natural growth hormone or in animalsused for food or wool production where the stimulation of growth hormonewill result in a larger, more productive animal.

DESCRIPTION OF THE INVENTION

The present invention is directed to a novel process for the preparationof the compound N- 1(R)- (1,2-dihydro-1-methanesulfonyl-spiro 3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methyl-propanamidewhich has the structure: ##STR4## and salts thereof, in particular, themethanesulfonate salt.

The instant process provides the desired compound from readily availableinexpensive and environmentally acceptable starting materials reagentsand solvents. The process does not require the use any chromatographicpurifications, and it is possible to produce the final product from theintermediate spiroindoline sulfonamide without isolation of any of theintermediates.

The individual processes within the general process are summarized asfollows: ##STR5##

(wherein L is an appropriate amino protecting group and X- is anappropriate counterion).

Within this general process, a first process concerns the preparation ofa compound of formula I: ##STR6## wherein L is an amino protectinggroup, by coupling an amino acid of the formula: ##STR7## with acompound of the formula: ##STR8## in the presence of an acid activatingagent in an inert solvent in the presence of a catalytic agent, to givethe compound of formula I.

Acid activating agents suitable for this process include: DCC, EDC, ECACand BOP, in which the preferred acid activating agent isDCC(N,N'-dicyclohexylcarbodiimide).

Catalytic agents suitable for this process include: HOBT and the like inwhich a preferred catalytic agent is HOBT (hydroxybenzotriazole).

Inert solvents appropriate for this processes include: acetonitrile;iso-propyl acetate; ethyl acetate; propionitrile; water; chlorinatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,dichloroethane, chlorobenzene, ortho-dichlorobenzene; benzene; toluene;xylenes; and the like; and mixtures thereof, in which the preferredsolvent is either acetonitrile or isopropyl acetate and water.

The preferred reaction temperature range is between -40° and 150° C.,and the most preferred range is between 20° and 35° C.

Suitable amino protecting groups include: benzyl, benzyloxymethyl,benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl,2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl, 2-chloro-benzyloxycarbonyl,2-chloroethyloxycarbonyl, di-t-amyloxycarbonyl,9-fluoroenylmethyloxycarbonyl, isopropoxycarbonyl,4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,2-nitrophenyl-sulfonyl, phthaloyl, 2,2,2-trichloro-t-butyloxycarbonyl,trifluoroacetyl, triphenylmethane, allyloxycarbonyl, andvinyloxycarbonyl groups, and the like, in which the preferred onesinclude benzyloxycarbonyl (carbobenzyloxy), t-butoxy-carbonyl groups,and in which the most preferred one is the t-butoxy-carbonyl group.

In the interest of efficiency, it is preferred that this coupling beconducted in situ without isolation of the compound of formula Ifollowing its preparation by the aforementioned process.

Within this general process, a second process concerns the preparationof a compound of formula II: ##STR9## which comprises reacting acompound of the formula I: ##STR10## wherein L is an amino protectinggroup, with an amino deprotecting agent to give the compound of formulaII.

Suitable amino protecting groups include: benzyl, benzyloxymethyl,benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl,2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl,2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl,di-t-amyloxycarbonyl, 9-fluoroenyl-methyloxycarbonyl,isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl,2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane,allyloxycarbonyl, and vinyloxycarbonyl groups, and the like, in whichthe preferred ones include benzyloxycarbonyI (carbobenzyloxy),t-butoxy-carbonyl groups, and in which the most preferred one is thet-butoxy-carbonyl group.

In this process, the removal of the amino protecting group may beaccomplished by use of an appropriate catalytic agent. Removal of at-butoxycarbonyl protecting group may be carried out in a solvent suchas methanol, ethanol, methylene chloride, ethyl acetate, or iso-propylacetate, with a strong acid. Such strong acids include methanesulfonicacid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride gas,hydrogen bromide; hydrogen iodide; trifluoromethane-sulfonic acid;camphorsulfonic acid; sulfuric acid; phosphoric acid; and anarylsulfonic acid, such as benzenesulfonic acid, p-toluenesulfonic acid,and p-chlorobenzene-sulfonic acid. Preferred catalytic agents include:trifluoroacetic acid; methanesulfonic acid; camphorsulfonic acid;benzenesulfonic acid, p-toluenesulfonic acid; andp-chlorobenzene-sulfonic acid. The most preferred catalytic agent ismethanesulfonic acid. The preferred solvent is methanol or ethanol, andthe most preferred solvent is ethanol.

The preferred reaction temperature range is between -40° and 150° C.,and the most preferred range is between 10° and 40° C.

Removal of a benzyloxycarbonyl (carbobenzyloxy) group may be achieved bya number of methods, for example, catalytic hydrogenation with hydrogenin the presence of a noble metal or its oxide such as palladium onactivated carbon in a protic solvent such as ethanol. In cases wherecatalytic hydrogenation is contraindicated by the presence of otherpotentially reactive functionality, the removal of benzyloxycarbonyl(carbobenzyloxy) group may also be achieved by treatment with a solutionof hydrogen bromide in acetic acid, or by treatment with a mixture ofTFA and dimethylsulfide.

In the interest of efficiency, it is preferred that this acid-catalyzeddeprotection be conducted in situ without isolation of the compound offormula II following its preparation by the aforementioned process.

Within this general process, a third process concerns the preparation ofa compound of formula III: ##STR11## wherein L is an amino protectinggroup, by coupling an amino acid of the formula: ##STR12## wherein L isan amino protecting group, with a compound of the formula II: ##STR13##in the presence of an acid activating agent in an inert solvent in thepresence of a catalytic agent, to give the compound of formula III.

Acid activating agents suitable for this process include: DCC, EDC, ECACand BOP, in which the preferred acid activating agent is DCC(N,N'-dicyclohexylcarbodiimide).

Catalytic agents suitable for this process include: HOBT and the like inwhich a preferred catalytic agent is HOBT (hydroxybenzotriazole).

Inert solvents appropriate for this processes include: acetonitrile;isopropyl acetate; ethyl acetate; propionitrile; water; chlorinatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,dichloroethane, chlorobenzene, ortho-dichlorobenzene; benzene; toluene;xylenes; and the like; and mixtures thereof, in which the preferredsolvent is a mixture of iso-propyl acetate and water, preferably in aratio of approximately 40:60 to 60:40 (by volume) and more preferably ina ratio of approximately 50:50 (by volume).

The preferred reaction temperature range is between -40° and 150° C.,and the most preferred range is between 20° and 50° C.

Suitable amino protecting groups include: benzyl, benzyloxymethyl,benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl,2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl,2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl,di-t-amyloxycarbonyl, 9-fluoroenyl-methyloxycarbonyl,isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl,2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane,allyloxycarbonyl, and vinyloxycarbonyl groups, and the like, in whichthe preferred ones include benzyloxycarbonyl (carbobenzyloxy),t-butoxy-carbonyl groups, and in which the most preferred one is thet-butoxy-carbonyl group.

In the interest of efficiency, it is preferred that this coupling beconducted in situ without isolation of the compound of formula IIIfollowing its preparation by the aforementioned process. Alternatively,the compound of formula III may be isolated as a discrete intermediate.

Within this general process, a fourth process concerns the preparationof a compound of formula IV, or a pharmaceutically acceptable saltthereof: ##STR14## which comprises reacting a compound of the formulaIII: ##STR15## wherein L is an amino protecting group, with an aminodeprotecting agent to give the compound of formula IV.

Suitable amino protecting groups include: benzyl, benzyloxymethyl,benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl,2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl,2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl,di-t-amyloxycarbonyl, 9-fluoroenyl-methyloxycarbonyl,isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl,2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane,allyloxycarbonyl, and vinyloxycarbonyl groups, and the like, in whichthe preferred ones include benzyloxycarbonyl (carbobenzyloxy),t-butoxy-carbonyl groups, and in which the most preferred one is thet-butoxy-carbonyl group.

In this process, the removal of the amino protecting group may beaccomplished by use of an appropriate catalytic agent. Removal of at-butoxycarbonyl protecting group may be carded out in a solvent such asmethanol, ethanol, methylene chloride, ethyl acetate, or iso-propylacetate, with a strong acid. Such strong acids include methanesulfonicacid, trifluoroacetic acid, hydrochloric acid, hydrogen chloride gas,hydrogen bromide; hydrogen iodide; trifluoromethane-sulfonic acid;camphorsulfonic acid; sulfuric acid; phosphoric acid; and anarylsulfonic acid, such as benzenesulfonic acid, p-toluenesulfonic acid,and p-chlorobenzene-sulfonic acid. Preferred catalytic agents include:trifluoroacetic acid; methanesulfonic acid; camphorsulfonic acid;benzenesulfonic acid, p-toluenesulfonic acid; andp-chlorobenzene-sulfonic acid. The most preferred catalytic agent ismethanesulfonic acid. It is preferred that compound of formula V isisolated in the form of the methanesulfonate salt. The preferred solventis methanol or ethanol, and the most preferred solvent is ethanol.

The preferred reaction temperature range is between -40° and 150° C.,and the most preferred range is between 10° and 40° C.

Removal of a benzyloxycarbonyl (carbobenzyloxy) group may be achieved bya number of methods, for example, catalytic hydrogenation with hydrogenin the presence of a noble metal or its oxide such as palladium onactivated carbon in a protic solvent such as ethanol. In cases wherecatalytic hydrogenation is contraindicated by the presence of otherpotentially reactive functionality, the removal of benzyloxycarbonyl(carbobenzyloxy) group may also be achieved by treatment with a solutionof hydrogen bromide in acetic acid, or by treatment with a mixture ofTFA and dimethylsulfide.

In the interest of efficiency, it is preferred that this acid-catalyzeddeprotection be conducted in situ without isolation of the compound offormula IV following its preparation by the aforementioned process.

Within this general process, a fifth process concerns the preparation ofa pharmaceutically acceptable salt of a compound of formula IV, inparticular, the methanesulfonate salt, i.e. a compound of formula V:##STR16## which comprises reacting a compound of the formula IV:##STR17## with in acid, preferably methanesulfonate acid, to give thecompound of formula V.

It is preferred that compound of formula V is isolated in the form ofthe methanesulfonate salt. The preferred solvent comprises ethyl acetateand ethanol, and the most preferrred solvent is a mixture of ethylacetate and ethanol.

In the interest of efficiency, it is preferred that the formation of thesalt be conducted in situ without isolation of the compound of formula Vfollowing its preparation by the aforementioned process.

In a preferred embodiment of the present invention, the individualprocesses within the general process are outlined as follows: ##STR18##

In this preferred embodiment, the CBZ-Spiroindoline 1 is treated withDarco (20% by weight) prior to hydrogenation. The hydrogenation iscarried out in ethanol at 65° C. over 10% Pd/C with vigorous stirring.

A solution of 1b in isopropyl acetate and water is coupled withcommercially available N-BOC-O-benzyl-D-serine in the presence ofdicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt). Afterfiltration of the dicyclohexylurea (DCU) side product, the 2-phasefiltrate is separated and the organic layer is washed successively with1M aqueous sodium hydroxide solution, 0.5M aqueous hydrochloric acid andfinally saturated aqueous sodium hydrogen carbonate. Improved results inthis coupling are achieved when a solution of the free amino iniPrOAc/H₂ O is treated with DCC, HOBT followed by addition of the aminoacid at ambient temperature and followed by reaction for 3-5 hrs Thebatch is then concentrated in vacuo and the solvent is switched fromisopropyl acetate to ethanol. This solvent switch generally proceedsswiftly by "feeding and bleeding" 3× batch volumes to remove isopropylacetate.

The BOC-group of 11 is removed by treatment with methanesulfonic acid(MsOH) (3 eq) in ethanol at 35°-40° C. Partitioning between isopropylacetate and aqueous 1M sodium hydroxide solution affords 12.

The coupling of 12 with N-BOC-α-aminoisobutyric acid is best conductedin a two-phase solvent system, isopropyl acetate/water (1:1) in thepresence of DCC and HOBt (1.1 eq. each). Removal of the DCU byfiltration, separation of the layers and washing the organic layersuccessively with 1M aqueous sodium hydroxide, 0.5M aqueous hydrochloricacid and saturated aqueous sodium hydrogen carbonate affords 14.

The mixture is solvent switched to ethanol for the subsequentmethanesulfonic acid cleavage of the Boc group. Deprotection of 14 ismore difficult than that of 11 and requires a concentrated solution ofethanol/methanesulfonic acid and heating to 35°-40° C. After extractiveworkup (EtOAc-NaOH), the free amine 15 is isolated. The organic layer iswashed well with 1N NaOH to ensure complete removal of methanesulfonicacid.

The ethyl acetate solution of the free base 15 is concentrated to lowbulk in vacuo and is azeotroped dry (KF <500 mg ml⁻¹) by "feeding andbleeding" 2× batch volumes of 90/10, ethyl acetate/ethanol followed by2× batch volumes of ethyl acetate. The resulting dry, slightly hazysolution of the free base 15 in ethyl acetate is treated with Darco G-60(25 weight %) at room temperature for about 10 hours. Removal of theDarco by filtration with a filtration agent gives the free base 15.

Formation of the methanesulfonic acid salt 16 from 15 is carried out inEtOAc with 1.1 eq of MsOH at about 50° C. The free base 15 is treatedwith 8 volume % of EtOH and 1 eq of H₂ O and heated to 55° C. untilcomplete dissolution. Cooling to ambient temperature and stirring theresulting slurry for 4 hours gives crystalline material of 16 designatedas crystal Form II solubility in IPA=12 mg/mL!.

The conversion of Form II to Form I is accomplished where the salt isformed in EtOAc-EtOH as above, but instead of cooling the initialsolution of the salt (at 55° C.) to ambient temperature, it is cooled to45° C. Crystals should start appearing at that temperature and theslurry should become thicker with time. The temperature is then raisedto 51° C. and the slurry is aged overnight. Complete conversion to FormI of 16 should be expected.

Preferably, the conversion of Form II to Form I is achieved by addingseed crystals of Form I to a solution of the free base in EtOAc-EtOH at50°-55° C. followed by aging. Accordingly, the free base 15 may betreated with 1.1 equivs. of methanesulfonic acid in 8% ethanol in ethylacetate at 50°-55° C. The batch is then seeded with approximately 2% byweight of Form I of the methanesulfonate salt 16, and then aged at 55°C. overnight. The batch is cooled to room temperature and aged forapproximately 2-3 hours. The product is isolated by filtration at roomtemperature under a nitrogen atmosphere, dried at 35° C. in vacuo andsieved to give the methanesulfonate salt 16.

The methanesulfonic acid salt 16 may also be formed by alternating thestepwise addition of MsOH (1.1 eq) and seed crystals of Form I to asolution of the free base in EtOAc-EtOH at about 50° C., wherein theorder of addition of the MsOH and the seed is not critical.

Throughout the instant application, the following abbreviations are usedwith the following meanings:

    ______________________________________                                        Bu       butyl                                                                Bn       benzyl                                                               BOC, Boc t-butyloxycarbonyl                                                   BOP      Benzotriazol-1-yloxy tris(dimethylamino)-                                     phosphonium hexafluorophosphate                                      calc.    calculated                                                           CBZ, Cbz Benzyloxycarbonyl                                                    DCC      N,N'-Dicyclohexylcarbodiimide                                        DIEA     Di-isopropylethylamine                                               DMF      N,N-dimethylformamide                                                DMAP     4-Dimethylaminopyridine                                              EDC      1-(3-dimethylaminopropyl)-3-ethylcarbodiimide                                 hydrochloride                                                        EDAC     Ethyl-3-(3-dimethylamino)-propylcarbodiimide                         EI-MS    Electron ion-mass spectroscopy                                       Et       ethyl                                                                eq.      equivalent(s)                                                        FAB-MS   Fast atom bombardment-mass spectroscopy                              h, hr.   hours                                                                HOBT, HOBt                                                                             Hydroxybenzotriazole                                                 HPLC     High pressure liquid chromatography                                  iPrOAc   iso-Propyl acetate                                                   KHMDS    Potassium bis(trimethylsilyl)amide                                   LAH      Lithium aluminum hydride                                             LHMDS    Lithium bis(trimethylsilyl)amide                                     Me       methyl                                                               MF       Molecular formula                                                    MHz      Megahertz                                                            MPLC     Medium pressure liquid chromatography                                MsOH     Methane sulfonic acid                                                NMM      N-Methylmorpholine                                                   NMR      Nuclear Magnetic Resonance                                           Ph       phenyl                                                               Pr       propyl                                                               prep.    prepared                                                             TFA      Trifluoroacetic acid                                                 THF      Tetrahydrofuran                                                      TLC      Thin layer chromatography                                            TMS      Tetramethylsilane                                                    ______________________________________                                    

In the above structural formula and throughout the instantspecification, the following terms have the indicated meanings:

The phrase "peptide coupling reaction" as used herein is intended tomean the coupling of a carboxylic acid with an amine using an acidactivating agent such as EDC, DCC, and BOP in an inert solvent in thepresence of a catalyst such as HOBT. Inert solvents appropriate for suchcouplings include: acetonitrile; iso-propyl acetate; ethyl acetate;propionitrile; water; chlorinated hydrocarbons such as dichloromethane,chloroform, carbon tetrachloride, dichloroethane, chlorobenzene,ortho-dichlorobenzene; benzene; toluene; xylenes; and combinationsthereof; and the like.

The variable "L" and the term "amino protecting group" is intended toindicate the presence of an appropriate protecting group for amino, suchas those described in Greene, T. W., Wuts, P. G. M. Protective Groups inOrganic Synthesis, 2nd ed., John Wiley & Sons, Inc., New York, 1991. Anappropriate protecting group will be able to withstand the reactionconditions of intermediate processes, prior to being removed whendesired. The amino protecting group is independently selected for eachprocess within the entire processes.

Suitable amino protecting groups include: benzyl, benzyloxymethyl,benzyloxycarbonyl (carbobenzyloxy), benzylsulfonyl,2-bromo-ethyloxycarbonyl, t-butoxy-carbonyl,2-chloro-benzyloxy-carbonyl, 2-chloroethyloxycarbonyl,di-t-amyloxycarbonyl, 9-fluoroenyl-methyloxycarbonyl,isopropoxycarbonyl, 4-methoxy-benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrophenyl-sulfonyl, phthaloyl,2,2,2-trichloro-t-butyloxycarbonyl, trifluoroacetyl, triphenylmethane,and vinyloxycarbonyl groups, and the like, in which the preferred onesinclude benzyloxycarbonyl (carbobenzyloxy), t-butoxy-carbonyl groups,and in which the most preferred one is the t-butoxy-carbonyl group.

The removal of the amino protecting group may be accomplished by use ofan appropriate catalytic agent. Removal of a t-butoxycarbonyl protectinggroup may be carded out in a solvent such as methanol, ethanol,methylene chloride, ethyl acetate, or iso-propyl acetate, with a strongacid. Such strong acids include methanesulfonic acid, trifluoroaceticacid, hydrochloric acid, hydrogen chloride gas, hydrogen bromide;hydrogen iodide; trifluoromethane-sulfonic acid; camphorsulfonic acid;sulfuric acid; phosphoric acid; and arylsulfonic acids, such asbenzenesulfonic acid, p-toluenesulfonic acid, andp-chlorobenzene-sulfonic acid. Preferred catalytic agents include:trifluoroacetic acid; methanesulfonic acid; camphorsulfonic acid;benzenesulfonic acid, p-toluenesulfonic acid; andp-chlorobenzene-sulfonic acid. The most preferred catalytic agent ismethanesulfonic acid. The preferred solvent is methanol or ethanol.

Removal of a benzyloxycarbonyl (carbobenzyloxy) protecting group may beachieved by a number of methods, for example, catalytic hydrogenationwith hydrogen in the presence of a noble metal or its oxide such aspalladium on activated carbon in a protic solvent such as ethanol. Incases where catalytic hydrogenation is contraindicated by the presenceof other potentially reactive functionality, the removal ofbenzyloxycarbonyl (carbobenzyloxy) group may also be achieved bytreatment with a solution of hydrogen bromide in acetic acid, or bytreatment with a mixture of TFA and dimethylsulfide.

The amine compounds employed as starting materials for the process ofthe present invention may be present as their acid salts, such as thesalts derived from using inorganic and organic acids. Examples of suchacids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic,trifluoroacetic, propionic, maleic, succinic, malonic, methane sulfonicand the like. Similarly the compounds produced by the processes of theinstant invention may be isolated in the form of their pharmaceuticallyacceptable acid salts. In addition, certain compounds containing anacidic function such as a carboxy can be in the form of their inorganicsalt in which the counterion can be selected from sodium, potassium,lithium, calcium, magnesium and the like, as well as from organic bases.

The preparation of compounds with the process of the present inventionmay be carried out in sequential or convergent synthetic routes. It isnoted that in some cases the order of carrying out the foregoingreaction schemes may be varied to facilitate the reaction or to avoidunwanted reaction products. In general, the process of the presentinvention is conducted in a sequential manner as presented herein.

Many of the starting materials are either commercially available orknown in the literature and others can be prepared following literaturemethods described for analogous compounds. The skills required incarrying out the reaction and purification of the resulting reactionproducts are known to those in the art. Purification procedures includecrystallization, normal phase or reverse phase chromatography.

The following examples are provided for the purpose of furtherillustration only and are not intended to be limitations on thedisclosed invention.

EXAMPLE 1 ##STR19## Isonipecotic acid-N-benzyl carbamate (3)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid (2) T.C.I.                                                                        4.02 kg (31.1 mol)                                      Benzyl chloroformate (Schweitzerhall)                                                               6.91 kg (40.5 mol)                                      K.sub.2 CO.sub.3      10.1 kg (72.9 mol)                                      Water                 40.2 L                                                  ______________________________________                                    

Isonipecotic acid (2) and K₂ CO₃ were dissolved in 40.2 L of water in a100 L 4 neck flask with mechanical stirring under N₂ and the solutionwas cooled to 10° C. Benzyl chloroformate was added, maintaining thetemperature between 9° and 14° C., and the mixture was warmed up to 22°C. after the addition was complete and aged for 58 h. The addition wascompleted in 4 h at which point the pH was 9.0. After aging for 58 hthere was no change in the pH.

The reaction mixture was transferred to a 200 L extractor and washedwith 3×13 kg (15 L) of IPAC and 1×12 L of EtOAc. The aqueous layer wasextracted with 8 L of toluene. After the washes the benzyl alcoholcontent was reduced from 3.8% to 1.4% by HPLC analysis. HPLC analytical:Dupont Zorbax 25 cm RXC8 column with 1.5 mL/min flow and detection at254 nm; isocratic mixture with 35% MeCN, 65% of 0.1% aqueous H₃ PO₄ ;retention times: 3=6.9 min, benzyl alcohol=3.3 min, toluene=17.3 min.

The aqueous phase was acidified with 37% aqueous HCl to pH 1.8. Carbondioxide was evolved during the addition of HCl, but gas evolution waseasily controlled. The addition of HCl took <1 h and required 10 L ofconc. HCl. The aqueous phase was extracted with 3×6.6 L of toluene. Thetoluene extracts were dried with 2 kg of sodium sulfate and filteredthrough a pad of Solka-floc™. The combined filtrates weighed 17.8 kg.The crude yield of carbamate 3 was 7.89 kg (97%) (as obtained byevaporation of weighed aliquots of the filtrates to dryness). Thefiltrates were transferred through a 10μ inline filter to a 100 L flask.The extracts were concentrated at 10 mbar at <25° C. to a volume of 18L. The final concentration of carbamate 3 was 440 g/L. The concentrationof the toluene filtrate served to azeotropically remove final traces ofwater (final KF=170 mg/L). The product was 99.1 area % pure with 0.9area % benzyl alcohol as the only impurity.

EXAMPLE 2 ##STR20## Isonipecotic acid chloride-N-benzyl carbamate (4)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid N-benzyl carbamate (3)                                                             7.89 kg (30.0 mol) in                                  in toluene. (MW = 263.30)                                                                            17.9 L                                                 Oxalyl chloride (MW = 126.93)                                                                        3.94 kg (31.0 mol)                                     DMF (MW = 73.10)         10 mL                                                Toluene                  12 L                                                 ______________________________________                                    

To the toluene solution of benzyl carbamate 3 from the preceding stepwas added 5 mL of DMF and 10 L of toluene. The oxalyl chloride was addedover a period of 20 min. The reaction mixture was aged for 16 h at 18°C. under a slow stream of nitrogen. HPLC analysis of the reactionmixture showed that 1.3% of the carboxylic acid 3 still remainedunreacted. The reaction mixture was warmed to 26° C., and 5 mL of DMFwere added. The mixture was aged for 2.5 h.

A 1.0 mL aliquot of the reaction mixture was quenched with 5.0 mL oftert-butylamine and analyzed after evaporation by HPLC: 25 cm DupontZorbax RXC8 column at 50° C. with 1 mL/min flow and detection at 220 nm;isocratic 42% MeCN, 58% of 0.1% aqueous H₃ PO₄. This method showed that<0.05% of the acid 3 remained (as Judged by A) and showed >3 area % B(>1 mol % (COCl)₂). ##STR21##

The mixture was concentrated at 10 mbar and a temperature of 20°-25° C.until 5 L of solvent had been removed.

The typical HPLC profile of concentrated toluene solution after t-BuNH₂quench described above is as follows:

    ______________________________________                                        Retention time (min)                                                                         Area %  Identity                                               ______________________________________                                        2.1            <0.5%   carboxylic acid 3                                      7.8            <0.5%   benzyl chloride                                        11.0           >99%    Cbz-t-butylcarboxamide A                               12.1           NA      toluene                                                12.7           <0.5%   ditert-butyloxamide B                                  ______________________________________                                    

EXAMPLE 3 ##STR22## Piperidine-4-carboxaldehyde-1-benzyl carbamate (5)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid chloride N-benzyl carbamate (4)                                                    3.38 kg (12.0 mol)                                     in toluene (MW = 281.74)                                                                             in 5.54 kg                                             DIEA (KF = 18 mg/L)    1.55 kg (15.0 mol)                                     10% Pd/C (KF < 20 mg/g)                                                                              101 g                                                  thioanisole (MW = 124.21, d = 1.058)                                                                 0.56 g                                                 ______________________________________                                    

The DIEA and thioanisole were added to the solution of (4) in toluenefrom the previous step and the catalyst was suspended in this mixture.The mixture was immediately placed into the 5 gal autoclave andhydrogenated at 20° C. and 40 psi of H₂. After 18 h the reaction hadtaken up 70% the theoretical mount of hydrogen and HPLC analysis of analiquot that was quenched with tert-butylamine indicated that 14.2 area% of acid chloride 2 remained. HPLC conditions same as above. Retentiontime: 5=8.1 min.

A second charge of catalyst (101 g) and thioanisole (0.54 g) were addedas a slurry in 1375 mL toluene to the hydrogenator. After 23 h HPLCanalysis of an aliquot that was quenched with tert-butylamine indicatedthat 1.8 area % of acid chloride 2 remained. The mixture was purged withnitrogen and the catalyst and precipitated DIEA•HCl were removed byfiltration through Solka-floc™. The filter cake was washed with 10 L oftoluene. The filtrates were transferred through a 10μ inline filter to a50 L extractor and washed with 2×7.2 L of 1M aqueous HCl and 2×7.2 L ofwater. The mixture was concentrated at 10 mbar and a temperature of25°-30° C. until 5 L of residue remained.

    ______________________________________                                        Retention time (min)                                                                         Area %      Identity                                           ______________________________________                                        2.1            <2          carboxylic acid 3                                  6.6            <1          dimer 21                                           8.1            >95         aldehyde 5                                         ______________________________________                                    

The assay yield of aldehyde 3 was 94% by HPLC analysis.

EXAMPLE 4 ##STR23## CBZ-Spiroindoline (9)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                              1.71 kg (6.89 mol)                                        carbamate (5) in toluene solution                                                                 in 21.4 kg                                                Phenylhydrazine      900 mL, 981 g (9.15 mol)                                 Trifluoroacetic acid (TFA)                                                                        2.20 L, 3.26 kg (28.6 mol)                                NaBH.sub.4          300 g, (7.93 mol)                                         Toluene             34.4 kg                                                   MeCN                7.0 L                                                     MeOH                7.0 L                                                     ______________________________________                                    

The crude aldehyde 5 solution from the previous step was transferredthrough a 10μ inline filter to a 100 L reactor equipped with Tefloncoated copper coils for cooling or heating and a mechanical stirrer.Toluene (34.4 kg) and MeCN (7 L) were added, and the resulting solutionwas cooled to 0° C. Phenylhydrazine was added in portions and thetemperature was maintained at -1° to 3° C. while nitrogen wascontinuously bubbled through the reaction mixture.

The phenylhydrazine was added until TLC and HPLC analysis indicatedcomplete consumption of the aldehyde 5 and the appearance of a slightexcess (<5%) of phenylhydrazine. TLC conditions: Silica, E. MerckKieselgel G60 F254 0.25 mm; diethyl ether/pentane (4/1); and developingagent 0.5% ceric sulfate, 14% ammonium molybdate in 10% aqueous sulfuricacid then heat; R_(f) : aldehyde 5=0.52, phenylhydrazone 7=0.61,phenylhydrazine 6=0.21. HPLC conditions: 25 cm Dupont Zorbax RXC8 columnat 30° C. with 1.0 mL/min flow and detection at 254 nm; gradientschedule:

    ______________________________________                                        Time (min)    acetonitrile:water                                              ______________________________________                                        0             57:43                                                           10            65:35                                                           15            75:25                                                           18            75:25                                                           ______________________________________                                    

retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min,phenylhydrazone 7=11.4 min.

The reaction mixture was aged for 30 min at 0°-2° C., and TFA was addedmaintaining the temperature between 2° and 7° C. The reaction mixturewas warmed to 50° C. over 30 min, and maintained for 17 h. The nitrogensparge through the reaction mixture was stopped and a slow stream ofnitrogen was maintained over the reaction mixture. During the first hourat 5° C. the color gradually darkened to a deep green, and a relativelysmall amount of a white crystalline precipitate (ammoniumtrifluoroacetate) formed. After 17 h HPLC analysis. (same conditions asabove) indicated that the reaction mixture contained 91.6 area %indolenine 8 and 1.5% of unreacted phenylhydrazone remained. Aging themixture for longer periods of time did not increase the assay yield ofindolenine 8.

The reaction mixture was cooled to 12° C., and 7.0 L of MeOH was added.NaBH₄ was added in small (<20 g) portions maintaining the temperaturebelow 15° C. The addition took 30 min. Moderate hydrogen evolution wasobserved during the addition, but it was easily controlled and there wasvirtually no frothing. Near the end of the addition the color rapidlychanged from green to brown and then bright orange. A small amount (<200mL) of a heavier phase had separated (presumably aqueous salts). HPLCanalysis (conditions as before) indicated that all of the indolenine 8had been consumed (90.4 area % CBZ-indoline 9); retention times:indolenine 8=7.5 min, indoline 9=8.2 min. TLC: ethyl ether as solvent,ceric sulfate-ammonium molybdate stain or 1% anisaldehyde stain;retention factors: indolenine 8=0.18, CBZ-indoline 9=0.33.

The color change from green to orange corresponds very closely toreaction end point. The quantity of NaBH₄ required to complete thereaction is heavily dependent on the temperature and rate of addition ofNaBH₄, but the yield and quality of the product is virtually unaffectedprovided that the reaction is complete. The reaction mixture was cooledto 5° C. over a period of 30 min. Then 8 L of 3% aqueous NH₄ OH (8 L)were added to bring the pH of the aqueous phase to 7.4, the mixture wasagitated, and allowed to settle. The temperature rose to 15° C. Thecloudy yellow lower aqueous phase was separated. The organic phase waswashed with 4 L of 3% aqueous NH₄ OH, 2×4 L of water, and 2×4 L ofbrine. The weight of the organic phase after the washings was 53.5 kg,and the assay yield was 94%.

The washed toluene solution was combined with the washed organic phasesof two other similarly processed reactions. The total aldehyde used inthe three reactions was 5.06 kg, (20.5 mol). The total weight ofCBZ-indoline 9 assayed in the combined organic phases was 5.91 kg, (18.3mol, 90% assay yield). The combined organic phases were dried with 5 kgof sodium sulfate, treated with 250 g of Darco G60 carbon for 30 min,and filtered through Solka-floc™. The filtrates were vacuum concentratedat 10 mbar at <25° C. until the residue was near dryness. The solventswitch was completed by slowly bleeding in 30 L of IPAC andreconcentrating to 14 L at 200 mbar at 50°-60° C. The mixture was heatedto reflux in order to obtain a clear homogeneous deep orange solution. ¹H NMR analysis indicated that the solution contained ca. 6 mol % ofresidual toluene after solvent switch.

The solution was cooled to 68° C. and seeded with 4 g of crystallineCBZ-indoline 9. The solution was allowed to gradually cool to 26° C.over 6 h and aged for 9 h at 20°-26° C. The slurry was cooled to 2° C.over 1 h and aged at 2° C. for 1h. The product was isolated byfiltration, and the filter cake was washed with 2×2 L of 5° C. IPAC and2×2 L of 5° C. MTBE. The product was dried in the vacuum oven at 30° C.under a nitrogen bleed to give 4.37 kg (74%) of the title compound 9 asa light tan crystalline powder. HPLC analysis of the product indicated99.5 area % purity. The mother liquor (11 L) and the washes contained1.15 kg (19%) of additional product 9 and ca 3% of Cbz-isonipecotic acidphenylhydrazide (retention time=4.8 min).

EXAMPLE 5 ##STR24## CBZ-Spiroindoline-methanesulfonamide (1)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline (9)                                                                             1.69 kg (5.23 mol)                                        Methanesulfonyl chloride                                                                           599 g (5.23 mol)                                         Et.sub.3 N (KF = 151)                                                                              635 g (6.27 mol)                                         THF (KF = 41)         12 L                                                    ______________________________________                                    

A 22 L flask was charged with the solid CBZ-spiroindoline 9 and then11.5 L of THF and the Et₃ N were transferred into the flask through a10μ inline filter. The resulting homogenous solution was cooled to 0° C.A 1 L dropping funnel was charged with the methanesulfonyl chloride and500 mL of THF. The solution of the MsCl in THF was added to the reactionmixture maintaining the temperature between 0° and 4° C. The additiontook 5 h and was exothermic. A white precipitate, presumablytriethylammonium hydrochloride formed during the addition. HPLC analysisindicated that the reaction was complete at the end of the addition (9was undetectable).

HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flowand detection at 254 nm. Gradient Schedule:

    ______________________________________                                        Time (min)   0.1% aq. H.sub.3 PO.sub.4 :MeCN                                  ______________________________________                                        0            70:30                                                            3            70:30                                                            12           20:80                                                            25           20:80                                                            ______________________________________                                         Retention times: 9 = 7.6 min, 1 = 13.6 min.                              

After the addition was complete the reaction mixture was warmed to 18°C. and aged for 16 h. There was no change in the appearance of thereaction mixture, and HPLC profile between the end of the addition andafter the 16 h age. The reaction mixture was slowly transferred over 1hinto a vigorously stirred solution of 30 L of water and 200 mL of 37%aqueous HCl in a 50 L flask. The temperature in the 50 L flask rose from22° to 28° C. The product separated as a pale tan gummy solid whichchanged to a granular solid. The aqueous suspension was cooled to 22° C.and aged for 1 h. The suspension was filtered, and the filter cake waswashed with 2×4 L of MeOH/water (50/50). HPLC analysis indicated that<0.1% of the CBZ-Spiroindoline-methanesulfonamide1 was in the motherliquors.

The filter cake was washed with 4 L of MeOH/water (50/50) to which 50 mLof 28% aqueous NH₄ OH had been added. The filter cake was washed with2×4 L of MeOH/water (50/50), and the solid was dried in the vacuum ovenat 50° C. under a nitrogen bleed to give 2.03 kg (97%) of the titleproduct 1 as an off-white powder. HPLC analysis of the solids indicated93.7 area % 1.

EXAMPLE 6 ##STR25## Optional Procedure for Isolation of IntermediateCBZ-Spiroindolenine (8)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                                12.37 g (0.050 mol)                                     carbamate (5)                                                                 Phenylhydrazine       5.41 g (0.050 mol)                                      Trifluoroacetic acid (TFA)                                                                          11.56 mL, 17.10 g                                                             (0.150 mol)                                             Methylene chloride    500 mL                                                  ______________________________________                                    

The CBZ-aldehyde 5 was dissolved in dichloromethane in a 1 L flaskequipped with Teflon coated magnetic stirring bar. The resultingsolution was cooled to 0° C. Phenylhydrazine was added via a weighedsyringe over 5 min and the temperature was maintained at -1° to 3° C.while nitrogen was continuously bubbled through the reaction mixture.

TLC and HPLC analysis indicated complete consumption of the CBZ-aldehyde5 and the appearance of a slight excess (<2%) of phenylhydrazine. TLCconditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethylether/pentane (4/1); and developing agent 0.5% ceric sulfate, 14%ammonium molybdate in 10% aqueous sulfuric acid then heat; R_(f) :aldehyde 5=0.52, phenylhydrazone 7=0.61, phenylhydrazine 6=0.21. HPLCconditions: 25 cm Dupont Zorbax RXC8 column at 30° C. with 1.0 mL/minflow and detection at 254 nm; gradient schedule:

    ______________________________________                                        Time (min)    acetonitrile:water                                              ______________________________________                                        0             57:43                                                           10            65:35                                                           15            75:25                                                           18            75:25                                                           ______________________________________                                    

retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min,phenylhydrazone 7=11.4 min.

The reaction mixture was aged for 10 min at 0°-2° C., and TFA was addedby syringe maintaining the temperature between 2° and 7° C. The reactionmixture was warmed to 35° C. over 30 min, and maintained for 17 h. Thenitrogen sparge through the reaction mixture was stopped and a slowstream of nitrogen was maintained over the reaction mixture. During thefirst hour at 35° C. the color gradually darkened to a rosy pink then toa deep green, and a relatively small amount of a white crystallineprecipitate (ammonium trifluoroacetate) formed. After aging for 17 hHPLC analysis (same conditions as above) indicated that the reactionmixture contained 93 area % indolenine 8 and <0.5% of unreactedphenylhydrazone remained. Aging the mixture for longer periods of timedid not increase the assay yield of indolenine 8. The reaction mixturewas cooled to 10° C., and a mixture containing 60 mL 28-30% ammoniumhydroxide, 90 mL water and 150 g crushed ice was added with goodstirring. The color of the mixture changed to a salmon color. Theorganic phase was separated and washed twice with 400 mL water then 100mL saturated aqueous NaCl. The organic phase was dried over magnesiumsulfate and filtered through a plug of 5 g of silica. The filtrate wasevaporated to give 15.84 g (99%) of indolenine 8 as a pale orange oil.

EXAMPLE 7 ##STR26## Procedure for the Preparation ofCBZ-Spiroindoline-methanesulfonamide (1) without Isolation ofIntermediate CBZ-Spiroindoline (9) Step 1 CBZ-Spiroindoline (9)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                                 49.5 g (0.20 mol)                                      carbamate (5)                                                                 Phenylhydrazine (Aldrich)                                                                            23.7 g (0.22 mol)                                      Trifluoroacetic acid (TFA)                                                                           75.4 g (0.66 mol)                                      Toluene (KF < 250 mg/L)                                                                               654 mL                                                MeCN (KF < mg/L)       13.3 mL                                                NaBH.sub.4             11.3 g, (0.30 mol)                                     Toluene                  20 mL                                                MeOH                     50 mL                                                ______________________________________                                    

A 2% (by volume) solution of MeCN in toluene was made up using 654 mL oftoluene and 13.3 mL of MeCN. In a 2 L 3 neck flask equipped with amechanical stirrer 617 ml of the above solution were degassed by passinga fine stream of nitrogen through the solution for 5 min.Phenylhydrazine and TFA were added to the mixture while still degassing.

The CBZ-aldehyde 5 was dissolved in the rest of the solution preparedabove (50 mL) and degassed by bubbling nitrogen through the solutionwhile in the addition funnel. The solution in the flask was heated to35° C., and the aldehyde solution was slowly added to thephenylhydrazine-TFA over 2 h. The mixture was aged at 35° C. for 16h.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 50° C. with 1 mL/minflow and detection at 220 nm; isocratic 55% MeCN, 45% of 0.1% aqueous H₃PO₄. Typical HPLC profile after 16 h age:

    ______________________________________                                        Retention time (min)                                                                         Area %   Identity                                              ______________________________________                                        1.6            0.1-0.5  phenylhydrazine 6                                     4.1            <0.1     dimer 21                                              4.7            <0.1     aldehyde 5                                            5.0            NA       spiroindoline 9                                       6.3            NA       toluene                                               6.9            97       spiroindolenine 8                                     10.3           <0.2     phenylhydrazone 7                                                    2-3 tot. other impurities < 0.2% ea.                           ______________________________________                                    

The mixture was cooled to -10° C. and MeOH was added. A suspension ofsodium borohydride in 20 mL toluene was added in small portions (1 mL)over 30 min taking care that the temperature did not exceed -2° C.

    ______________________________________                                        Area %          Identity                                                      ______________________________________                                        0.1-1           phenylhydrazine 6                                             85-90           CBZ-spiroindoline 9                                           <0.1            CBZ-spiroindolenine 8                                         10-15 tot.      other impurities (<3% ea.)                                    ______________________________________                                    

The temperature was raised to 10° C. over 1h, and 6% aqueous ammonia(200 mL) was added. The mixture was agitated for 10 min, allowed tosettle for another 10 min, and the lower aqueous phase was drawn off.Acetonitrile (20 mL) and MeOH (20 mL) were added to the organic phaseand it was washed with 150 mL of 15% brine. The organic phase was foundto contain a 92% assay yield of CBZ-spiroindoline 9.

Step 2 CBZ-Spiroindoline-methanesulfonamide (1)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline (9) (MW = 322.51)                                                               (0.184 mol)                                               Methanesulfonyl chloride                                                                          21.1 g (0.184 mol)                                        DIEA (KF = 150 mg/L)                                                                              29.7 g, 40.1 mL (0.230 mol)                               THF (KF = 41 mg/L)  150 mL                                                    ______________________________________                                    

The crude solution of CBZ-spiroindoline 9 solution from Step 1 above wasconcentrated in a 1L 3 neck flask (60°-70° C., 150-200 Torr) until 250 gof residue remained. The THF and DIEA were added, and the resultinghomogenous solution was cooled to 0° C. A 125 mL dropping funnel wascharged with the methanesulfonyl chloride and 50 mL of THF. The solutionof MsCl in THF was added over 2 h to the reaction mixture maintainingthe temperature between 0° and 4° C. and the mixture was aged for 2 h at5°-8° C. The addition was slightly exothermic. A white precipitate,presumably DIEA-hydrochloride, formed during the addition. HPLCconditions were the same as above. HPLC analysis indicated that thereaction was complete 1 h after the end of the addition (9 wasundetectable) and the assay yield was 94% from 9. Retention time: 1=7.8min. Typical HPLC profile of reaction mixture after 2 h age:

    ______________________________________                                        Area %          Identity                                                      ______________________________________                                        <0.1            CBZ-spiroindoline 9                                           90-92           CBZ-sulfonamide 1                                             8-10 tot.       other impurities (<2% ea.)                                    ______________________________________                                    

The mixture was warmed to 20° C., and 200 mL of 1M aqueous HCl wasadded. The mixture was warmed to 50° C., and the aqueous phase wasseparated. The organic phase was washed sequentialy with 100 mL water,100 mL 5% aqueous sodium bicarbonate, and 100 mL water. The organicphase was transferred to a 1 L 3 neck flask equipped for mechanicalstirring and distillation. The mixture (ca 400 mL) was distilled atatmospheric pressure until 150 mL of distillate had been collected. Thehead temperature reached 107° C.; the pot temperature was 110° C. Thedistillation was continued with continuous addition of n-propanol atsuch a rate as to maintain a constant volume (ca 350 mL) in the pot. Thedistillation was stopped when a total of 525 mL of n-PrOH had been addedand a total of 800 mL of distillate had been collected.

The temperature of both the head and pot rose from 94° C. to 98° C.during the solvent switch. Toluene and n-PrOH form an azeotrope boilingat 97.2° C. composed of 47.5% toluene and 52.5% n-PrOH. The mixture wasallowed to cool gradually to 20° C. over 3h and aged for 12 h. Themother liquor was found to contain 2% toluene and 4 mg/mL ofsulfonamide. The solubility of the sulfonamide in various mixtures oftoluene and n-PrOH has been determined by HPLC assay:

    ______________________________________                                        % toluene in n-PrOH                                                                           solubility of 1 in mg/mL                                      ______________________________________                                        0               2.36                                                          5               3.02                                                          10              4.23                                                          20              7.51                                                          25              10.3                                                          ______________________________________                                    

The crystalline slurry was filtered and washed with 3×100 mL of n-PrOH.The product was dried in a vacuum oven at 50° C. with a nitrogen bleedfor 16 h to furnish 65.5 g (82% from aldehyde 5) of 6 as a tan solidwith 93.5 wt % purity.

Typical HPLC profile of solid:

    ______________________________________                                        Area %         Identity                                                       ______________________________________                                        <0.1           CBZ-spiroindoline 9                                            >99            CBZ-sulfonamide 1                                              <1 tot.        other impurities (<0.2% ea.)                                   ______________________________________                                    

For additional purification, a 40.0 g sample of the n-PrOH crystallizedsulfonamide was dissolved in 134 mL of EtOAc at 60° C. and treated with8.0 g of Darco G-60 carbon for 1 h at 60° C. After the addition of 2.0 gSolkafloc™, the slurry was filtered through a pad of 4.0 g Solkafloc™,and the pad was washed with 90 mL of EtOAc at 60° C. Prior to theaddition of the carbon the solution was a brown color. The filtrationproceeded well without plugging to give a golden yellow filtrate. Thefiltrate was distilled at atmospheric pressure in a 500 mL flask (pottemperature 80°-85° C.) until 100 g (100 mL) of residue remained. Thissolution was allowed to cool to 35° C. over 3 h. Over a 1h period, 116mL of cyclohexane was added with good agitation at 35° C. The mixturewas cooled to 20° C. over 1 h and aged at 20° C. for 12 h. At 35° C.much of the sulfonamide has crystallized out and the mixture was thick.Addition of cyclohexane at 20° C. makes agitation difficult. After theaging period, the supernatant was found to contain 2.5 mg 1/g. Thecrystalline slurry was filtered and the cake was washed with 77 mL of2:1 cyclohexane-EtOAc and 2×77 mL of cyclohexane. The product was driedin a vacuum oven at 50° C. with a nitrogen bleed for 16 h to furnish34.2 g of 1 (MW=400.3) as a white crystalline solid (85% recovery fromcrude 1, 70% from 5 with >99.9 wt % purity).

EXAMPLE 8 ##STR27## HCl Salt of Spiroindoline-methanesulfonamide (1a)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-spiroindoline-methanesulfonamide (1)                                                              941 g (2.35 mol)                                      Pearlman's catalyst 20% Pd(OH).sub.2 /C                                                               188 g                                                 THF                      8 L                                                  MeOH                     7 L                                                  ______________________________________                                    

The catalyst was suspended in 7 L of MeOH and transferred into the 5 galautoclave followed by the solution of 1 in 8 L of THF. The mixture washydrogenolyzed at 25° C. at 80 psi of H₂. After 2.5 h the temperaturewas raised to 35° C. over 30 min.

HPLC analysis indicated complete consumption ofCbz-spiroindoline-methanesulfonamide. HPLC conditions: 25 cm DupontZorbax RXC8 column with 1.5 mL/min flow and detection at 254 nm.

Gradient Schedule:

    ______________________________________                                        Time (min)   0.1% aq. H.sub.3 PO.sub.4 :MeCN                                  ______________________________________                                        0            70:30                                                            3            70:30                                                            12           20:80                                                            25           20:80                                                            ______________________________________                                         retention times: Spiroindoline = 7.6 min,                                     Cbzspiroindoline-methanesulfonamide = 13.6 min.                          

The mixture was purged with nitrogen and the catalyst was removed byfiltration through Solka-floc™ while still warm. The catalyst was washedwith 4 L of THF and 2 L of MeOH. The pale yellow filtrates wereconcentrated to a thick oil at 10 mbar and <25° C. The solvent switchwas completed by slowly bleeding in 15 L of EtOAc and reconcentrating todryness. The residue solidified to a hard off-white mass. MeOH (1.5 L)was added and the mixture was heated to 70° C. to give a homogenoussolution. While the solution was at 70° C., 10.5 L of EtOAc at 20° C.was added. The temperature fell to 40° C., and the mixture remainedhomogenous.

Subsequent experiments suggested that it is more convenient to solventswitch the MeOH-THF filtrates to MeOH, concentrate to the desiredvolume, and then add the EtOAc. This avoids the solidification of theresidue upon concentration of the EtOAc solution.

Hydrogen chloride diluted with about an equal volume of nitrogen waspassed into the solution. The temperature rose to 60° C. over the courseof 15 min, and a white precipitate of the hydrochloride salt formed.Diluting the HCl with nitrogen only avoids the reaction mixture suckingback and may not be necessary.

The mixture was cooled in an ice bath, and the hydrogen chlorideaddition was continued for 1h. The temperature gradually fell to 20° C.The suspension was aged for 2 h while the temperature was lowered to 10°C. The crystalline product was isolated by filtration, and the filtercake was washed with 3 L of EtOAc. It was dried in the vacuum oven at35° C. to give 1.18 kg (86%) of the title product 1a as an off-whitecrystalline solid of >99.5 area % purity by HPLC analysis. HPLCconditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow anddetection at 230 nm; isocratic 35% MeCN, 65% of 0.1% aqueous ammoniumacetate. Retention time: 1a=5.4 min.

EXAMPLE 9 ##STR28## Spiroindoline-methanesulfonamide (Free base form)(1b)

A 250 mL aliquot of the filtrate from the Cbz-hydrogenolysis containing4.67 g of 1b (free base) was concentrated to ca 10 mL. The residue wasdissolved in 20 mL of EtOAc and the solution was reconcentrated to ca 10mL. This was repeated once more, and 10 mL of EtOAc was added to theresidue. A crystalline precipitate began to form. MTBE (20 mL) was addedin one portion. Additional crystalline solid precipitated, but thesupernatent still contained a substantial quantity of dissolved productwhich did not precipitate on standing. Hexanes (70 mL) were addeddropwise over 2 h to the mixture with vigorous stirring. The slowaddition of the hexanes is neccessary to avoid the oiling out of theamine.

The agitated mixture was aged for 1 h and filtered. The filter cake waswashed with 20 mL of 1:1 MTBE-hexanes and then with 20 mL of hexanes.The product was dried under a stream of nitrogen to give 3.86 g (82%) ofthe free amine of 1b as an off white crystalline solid of >99.5 area %purity. HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/minflow and detection at 230 nm; isocratic 35% MeCN, 65% of 0.1% aqueousammonium acetate. Retention time: 1b=5.4 min.

EXAMPLE 10A ##STR29## Spiroindoline-methanesulfonamide (Free base form)(1b)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline-sulfonamide (1)                                                                   833.5 gr (2.08 mol)                                     Pd(OH).sub.2 /C (20% weight of Pd(OH).sub.2)                                                        124.5 (15%)                                             THF                    6.5 L                                                  MeOH                   19.5 L                                                 NH.sub.4 OH (conc)      60 mL                                                 ______________________________________                                    

The hydrogenation was run three (3) times due to equipment limitations;this procedure refers to a single run. The CBZ spiroindolinesulfonamide1 was dissolved in THF (6.5 L, KF=53 μg/μL) and then MeOH(KF=18 μg/mL, 4L) was added followed by addition of the catalyst and theslurry was transferred to a 5 gal autoclave. The remainder of the MeOH(2.5 L) was used for rinsing. The mixture was heated to 40° C. at 50 psifor 24 hours. The catalyst loading and reaction time are a function ofthe purity of starting material 1. This material was unique requiring≧15% catalyst and long reaction time. Purer batches of spiroindolinerequired only 5% of catalyst and 4-6 hrs reaction time.

Upon completion (<0.1 A % 1 by LC) the mixture was filtered thru SolkaFloc™ and the carbon cake washed with MeOH (13 L) containing NH₄ OH(0.5%, 60 mL). The combined filtrates (assay shows 1587 g ofspiroindoline amine 1b) were concentrated in vacuo and the resultingsolids were partitioned between 40 L (of toluene:THF (3:1) and 0.5N NaOH(18 L). Although the layers separated easily a heavy precipitate couldbe seen in the aqueous layer. The aqueous suspension was thus extractedwith CH₂ Cl₂ (15 L). The aqueous and organic layer separated slowly.Prior to CH₂ Cl₂ addition THF was added to the aqueous layer along withenough NaCl to saturate the layer. However dissolution of the productwas not achieved which necessitated the use of CH₂ Cl₂.

The combined toluene, THF and CH₂ Cl₂ layers were combined andconcentrated in the batch concentrator. The residue was flushed with 7 Lof CH₃ CN. Finally 10 L of CH₃ CN were added and the solution stoodovernight under N₂ atmosphere.

EXAMPLE 10B ##STR30## Spiroindoline-methanesulfonamide (Free base form)(1b)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline-sulfonamide (1)                                                                     3 kg (7.49 mol)                                       Darco G-60             600 g                                                  Ethyl Acetate           36 L                                                  Absolute Ethanol       189 L                                                  10% Ps/C               450 g                                                  Ammonia Solution       500 ml                                                 Solka Floc ™         2.5 kg                                                Isopropyl Acetate       65 L                                                  ______________________________________                                    

A mixture of CBZ-spiroindoline (1) (1 kg) and Darco G-60 (200 g) inethyl acetate (9 L) was stirred and heated at 60°-65° C. under anitrogen atmosphere for 8 hours. The Darco was removed by filtration at60°-65° C., the solid washed with hot ethyl acetate (3 L) and thefiltrate and washings combined. LC wt/wt assay confirmed negligible lossto the Darco. The ethyl acetate solution was evaporated to dryness invacuo using a 20 L Buchi apparatus and then flushed with absoluteethanol (2×5 L). This material was then slurried in absolute ethanol (8L) warmed to 65°-70° C. and placed in the 20 L autoclave. The batch wasrinsed in with absolute ethanol (1 L). A slurry of 10% Palladium oncharcoal (75 g, 7.5% by weight) in absolute ethanol (750 ml) was thenadded to the autoclave and rinsed in with a further portion of absoluteethanol (250 ml).

The batch was hydrogenated at 65° C. with vigorous stirring under 40 psihydrogen pressure for 3 hours, a second portion of 10% palladium oncharcoal (75 g) was added, the batch was hydrogenated for a further 2hours and then sealed overnight. The batch was transferred (still hot,60°-65° C.) to a 20 L Buchi apparatus and degassed in vacuo to removeformic acid by "feeding and bleeding" absolute ethanol (18 L total).

This procedure was repeated twice more and the three batches werecombined in a 10 gallon glass-lined vessel and the combined batch wasdegassed again by the addition and distillation (in vacuo) of absoluteethanol (2×10 L). Solka floc™ (0.5 kg) was added to the batch and rinsedin with ethanol (10 L). An Estrella filter was loaded with Solkafloc™ (2kg) as a slurry in ethanol (20 L). The resulting mixture was warmed to60°-65° C. and then transferred at this temperature via heated filterusing pump to two tared stainless-steel bins. The initial vessel, thefilter, the pump and the lines were rinsed with a hot (60°-65° C.)mixture of aqueous ammonia (500 ml) in absolute ethanol (25 L). Thefiltrate and washings were combined in the two stainless-steel bins.

The batch was then transferred to a vessel using an in-line filtercontaining a 10 micron cartridge, and then concentrated in vacuo to lowbulk (˜15 L). The ethanol was replaced by isopropyl acetate by the"feeding and bleeding" of 3× batch volumes of isopropyl acetate (45 Ltotal), while maintaining a batch volume of ˜15 L. The solvent switch,when complete, contained <1% residual ethanol by GC. The batch was thendiluted to ˜33 L by the addition of isopropyl acetate (20 L), and thissolution of spiroindoline-amine 1b (1.855 kg by LC analysis) inisopropyl acetate was used for the next stage of the process.

EXAMPLE 11A ##STR31## Boc-O-Benzylserine Spiroindoline (11)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline-amine (1b)                                                                          1587 g (5.966 moles)                                      Amino acid (10)     1938 g (6.563 moles)                                       ##STR32##                                                                    DCC                 1334.5 g (6.563 moles)                                    HOBT                884 g (6.563 moles)                                       CH.sub.3 CN         25 L                                                      0.5N NaOH           18 L                                                      0.5N HCl            18 L                                                      NaHCO.sub.3 sat.    18 L                                                      iPrOAc              28 L                                                      ______________________________________                                    

The spiroindoline-amine1b in CH₃ CN or iPrOAc:H₂ O (25 L) at ambienttemperature under N₂ was treated in sequence with HOBT (884 g; 1.1 eq)as a solid, DCC (1334.5 g, 1.1 eq) as the melt (heating in hot water at60° C. for ca. 1 hr) and finally the amino acid 10 (1938 g) as thesolid. The mixture was stirred for 3 hr upon which time heavyprecipitation of DCU occurred and LC analysis showed ca. 0.5 A % ofamine 1b remaining. IPAc (9 L) was added, the slurry was filteredthrough Solka Floc™ and the cake was washed with IPAc (19 L) . Thecombined organic solution was washed in sequence with 0.5N NaOH (18 L),0.5N HCl (18 L) and saturated NaHCO₃ (18 L). A final water wash at thispoint resulted in an emulsion and was thus eliminated.

The organic layer was concentrated in vacuo and the residue wasdissolved in MeOH or EtOH (10 L final volume). Assay yield 3026 gr(89%).

The use of alternative peptide coupling agents such ascarbonyldiimidazole or formation of mixed anhydrides, such as sec-butylcarbonate, gave inferior yields of 11 and/or 14 with a high degree ofepimerization in the case of the former compound. Other peptide couplingreagents were prohibitively expensive.

EXAMPLE 11B ##STR33## Boc-O-Benzylserine Spiroindoline (11)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline-amine (1b)                                                                             1.855 kg (6.96 mol)                                    isopropyl acetate      29 L                                                   Dicyclohexylcarbodiimide (DCC)                                                                       1.58 kg (7.65 mol)                                     1-Hydroxybenzotriazole (HOBt)                                                                        1.03 kg (7.62 mol)                                     N-Boc-O-benzyl-D-Serine                                                                              1.26 kg (7.65 mol)                                     1M Aqueous sodium hydroxide                                                                          26 L                                                   0.5M Aqueous hydrochloric acid                                                                       26 L                                                   Satd. Aqueous sodium hydrogen carbonate                                                              26 L                                                   Absolute Ethanol       50 L                                                   ______________________________________                                    

Water (20 L) was added to a stirred solution of thespiroindoline-amine1b (1.855 kg) in isopropyl acetate (33 L) in areaction vessel. The following chemicals were then added sequentially atroom temperature under a nitrogen atmosphere: DCC (1.58 kg, 1.1equivs.), HOBt (1.03 kg, 1.1. equivs.) and finallyN-Boc-O-benzyl-D-Serine (2.26 kg, 1.1 equivs.). The reagents were rinsedin with isopropyl acetate (7 L). The batch was stirred at roomtemperature under nitrogen atmosphere for 5 hours when LC showed theratio of product/starting material to be 99.4/0.6. The mixture was thenfiltered through an Estrella filter using cloth and cardboard only andutilizing a pump into another vessel. The sending vessel was rinsed withisopropyl acetate (22 L) and this was used to rinse the filter, the pumpand the lines into the receiving vessel. The 2-phase mixture in thevessel was stirred for 10 minutes and then allowed to settle for 15minutes. The lower aqueous layer was separated off and the organicsolution was left to stand at room temperature overnight.

The next day, the organic solution was washed with 1M aqueous sodiumhydroxide solution (26 L) then 0.5M aqueous hydrochloric acid (26 L) andfinally saturated aqueous sodium hydrogen carbonate (26 L). LC analysisgave an assay yield of 3.787 kg, 93% overall yield from 7.49 moles (3kg) of starting CBZ-spiroindoline (1). The batch was concentrated invacuo (internal temperature=13°-15° C. jacket temperature=40° C.,Vacuum=29") to low bulk (˜15 L) and solvent switched to ethanol by"feeding and bleeding" ethanol (50 L) whilst maintaining the volume at˜15 L. GC showed <1% isopropyl acetate remaining. This solution was usedfor the next stage of the process.

EXAMPLE 12A ##STR34## O-Benzylserine Spiroindoline (free base form) (12)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc-O-Benzylserine Spiroindoline (11)                                                               3026 g (5.57 moles)                                     Methane sulphonic acid (MsOH)                                                                       1.16 L (17.9 moles)                                     MeOH                  10 L                                                    iPrOAc                24 L                                                    0.5 N NaOH            35 L                                                    ______________________________________                                    

The Boc-O-benzylserine spiroindoline 11 in 10 L of MeOH (or EtOH) wastreated with neat MsOH (1.16 L) added over ca. 30-40 min, (initialtemperature 16° C., final temperature 28° C.). The dark red solution wasaged overnight under N₂. The mixture was then pumped into a 100 Lextractor containing 24 L iPrOAc and 35 L 0.5N NaOH. The pH of theaqueous layer was 7. NaOH (6M) was added until pH ≧10.5. As the pHincreased the color changed from red to yellow. The layers wereseparated and the organic layer (24 L) was shown by NMR to contain 13mole % of MeOH in iPrOAc 5 volume %!. LC assay 2.48 kg.

EXAMPLE 12B ##STR35## O-Benzylserine Spiroindoline (free base form) (12)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc-O-Benzylserine Spiroindoline (11)                                                               3.787 kg (6.96 mol)                                     Methanesulphonic acid 2.006 kg (20.87 mol)                                    Isopropyl acetate     38 L                                                    1M Aqueous sodium hydroxide                                                                         16 L                                                    50% Aqueous sodium hydroxide                                                                        1.6 L                                                   ______________________________________                                    

Methanesulphonic acid (2.006 kg, 1.355 L, ˜3 equivs.) was added to thestirred solution of Boc-O-benzylserine spiroindoline (11) (3.787 kg) inethanol (total volume ˜15 L) in a reaction vessel. The batch was warmedto 35°-40° C. After 7 hours, LC showed the absence of starting materialand the reaction was allowed to cool to room temperature overnight. Thenext day, water (44 L) was added to the batch with stirring. The batchwas cooled to ˜5°, stirred for 30 minutes and then filtered through anin-line filter (loaded with a 10μ cartridge) into a bin. The batch wasthen sucked back into the vessel. A water rinse (10 L) was used to rinsethe vessel and lines into the bin and this was used to then rinse backinto the vessel. Isopropyl acetate (38 L) was added followed by a 1Maqueous sodium hydroxide (16 L). The batch was cooled to 10°-15° C., thepH of the lower aqueous layer was confirmed as ˜7 and 50% aqueous sodiumhydroxide solution was added (1.6 L) (pH>10). The batch was stirred at10°-15° C. for 25 minutes and then allowed to settle for 10-15 minutes.The lower aqueous layer was separated (78.1 kg). LC assay indicated 28.4g of 12 (0.85% of theory) contained in the aqueous liquors. Volume ofthe organic solution=51 L. LC assay indicated 3.057 kg, 92% overallyield from 3 kg, 7.49 moles of CBZ-spiroindoline sulfonamide (1). Thissolution was used for the next stage.

EXAMPLE 13A ##STR36## Boc-Aminoisobutyryl O-Benzylserine Spiroindoline(14)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline-amine (12)                                                                          2481 g (5.57 moles)                                       amino acid peptide (13)                                                                           1247.1 g (6.16 moles)                                      ##STR37##                                                                    DCC                 1266.7 g (6.16 moles)                                     HOBT                827 g (6.16 moles)                                        IPAc                52 L                                                      H.sub.2 O.sup.-     37 L                                                      0.5N NaOH           36 L                                                      0.5N HCl            36 L                                                      Sat. NaHCO.sub.3    36 L                                                      ______________________________________                                    

The solution of the amine 12 in IPAc was diluted to a total volume of 39L with IPAc and 37 L of H₂ O was added. The biphasic mixture was thentreated in sequence with HOBT (827 g) as a solid, DCC (1266.7 g) as amelt, and amino acid 13 at ambient temperature under nitrogen. Thereaction mixture was stirred for 2 h upon which lime LC analysisindicated dissappearance of the starting material 12 (<0.3 A %). Themixture was filtered through Solka Floc™ and the solids were washed with13 L of IPAc. The material may be stored at this point as a biphasicmixture overnight.

The mixture was transferred to a 100 L extractor, the aqueous layer wasseparated and the organic layer was washed successively with 36 L of0.5N NaOH, 0.5N HCl and saturated NaHCO₃. Assay yield 3160 g (81% fromspiroindoline ±5% for volume measurement error). The solution wasconcentrated to a small volume and was flushed with ethanol (2×4 L) . Ifdesired, the immediate compound 14 may be isolated by adding water tocrystalize it out.

The use of alternative peptide coupling agents such ascarbonyldiimidazole or formation of mixed anhydrides, such as sec-butylcarbonate, gave inferior yields of 14 with a high degree ofepimerization. Other peptide coupling reagents were prohibitivelyexpensive.

EXAMPLE 13B ##STR38## Boc-Aminoisobutyryl O-Benzylserine Spiroindoline(14)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline amine (12)                                                                             3.057 kg (6.89 mol)                                    Dicyclohexylcarbodiimide (DCC)                                                                       1.56 kg (7.56 mol)                                     1-Hydroxybenzotriazole (HOBt)                                                                        1.02 kg (7.55 mol)                                     Boc-2-Aminoisobutyric acid (13)                                                                      1.54 kg (7.58 mol)                                     Isopropyl acetate      32 L                                                   1M Aqueous sodium hydroxide                                                                          38 L                                                   0.5M Aqueous hydrochloric acid                                                                       38 L                                                   Satd. aqueous sodium hydrogen carbonate                                                              38 L                                                   Absolute ethanol       45 L                                                   ______________________________________                                    

Water (49 L) was added to the stirred solution of the spiroindolineamine 12 (3.057 kg) in isopropyl acetate (total volume ˜51 L) in areaction vessel at room temperature under a nitrogen atmosphere. Thefollowing chemicals were then added sequentially: DCC (1.56 kg, ˜1.1equivs.), HOBt (1.02 kg, ˜1.1 equivs.) and finally,N-Boc-2-aminoisobutyric acid 13 (1.54 kg, ˜1.1 equivs.). The mixture wasstirred vigorously at room temperature for 2 hours when LC showed thereaction to be complete. The mixture was filtered to to another vesselvia an Estrella filter using a pump. Isopropyl acetate (22 L) was usedto rinse vessel, the filter, the pump and the lines into the receivingvessel. The 2-phase mixture was then stirred for 5 minutes and thelayers were allowed to separate. The lower aqueous layer was separatedwithout incident (weight of aqueous liquors=51.1 kg). The organicsolution was then washed sequentially with 1M aqueous sodium hydroxide(38 L), 0.5M aqueous hydrochloric acid (38 L) and finally, saturatedaqueous sodium hydrogen carbonate (38 L) without incident.

The organic solution was then transferred using a pump via an in-linefilter (containing a 10μ cartridge) to another vessel for the solventswitch to ethanol. The vessel was rinsed with isopropyl acetate (10 L)and this was used to rinse the pump, the filter and the lines into thereceiving vessel. The filtrate and washings were combined. Totalvolume=75 L (by dipstick). LC assay gave 4.395 kg of Boc-aminoisobutyrylO-benzylserine spiroindoline (14), i.e. 93% overall from 7.49 moles ofstarting CBZ-spiroindoline sulfonamide (1).

The batch was concentrated in vacuo to low bulk (˜15 L) and theisopropyl acetate switched to ethanol by "feeding and bleeding" absoluteethanol (45 L total). At the end of the solvent switch, GC showed <1%isopropyl acetate remaining. This solution (25 L) containing 4.395 kg of14 was used for the next stage. If desired, the inermediate compound 14may be isolated by adding water to crystalize it out.

EXAMPLE 14A ##STR39## Aminoisobutyryl O-Benzylserine Spiroindoline (15)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc Spiroindoline (14)                                                                             3160 g (5.03 moles)                                      Methanesulfonic acid (MsOH)                                                                         979 mL (15.1 moles)                                     EtOH                   6.2 L                                                  H.sub.2 O             30 L                                                    1N NaOH               11 L                                                    EtOAc                 26 L                                                    Darco 60 activated carbon                                                                            1 Kg                                                   ______________________________________                                    

The Boc spiroindoline 14 was dissolved in 6.2 L of EtOH and treated withMsOH (979 mL). The temperature rose from 20° to 30° C. and the reactionwas allowed to proceed overnight. After 12 hours at 20° C. there wasstill 15 A % of starting material left so the mixture was heated to 35°C. for 6 hours. Upon completion (<0.1 A % 14) the reaction was cooled to20° C. and 30 L of H₂ O were added and the solution was filtered througha glass funnel with a polypropylene filter to filter off residual DCU.The mixture was transferred to a 100 L extractor and 26 L of EtOAc wereadded. The aqueous layer was basified via addition of chilled 1N NaOH(11 L) and 1 L of 50% NaOH. Addition of ice was required to keep thetemperature below 14° C. Higher temperatures resulted in significantemulsion problems.

The organic layer was distilled at 50° C. at ca. 21" of Hg until KF<1000 μg/mL. Lower KF's result in more efficient carbon treatments andbetter recovery at the salt formation step. KF's of 160 μg/mL wereachieved at the 700 g scale. The solution was diluted with ethyl acetateto a total volume of 31 L (LC assay 2.40 kg). Activated carbon (DarcoG-60) was added and the mixture was stirred for 24 h. The mixture wasfiltered through Solka Floc™ and the filter cake was washed with ethylacetate (16 L), assay 2.34 Kg.

EXAMPLE 14B ##STR40## Aminoisobutyryl O-Benzylserine Spiroindoline (15)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc Spiroindoline (14)                                                                             4.395 kg (6.99 mol)                                      Methanesulfonic acid 2.017 kg (20.99 mol)                                     Ethyl acetate          185 L                                                  1M Aqueous sodium hydroxide                                                                          16 L                                                   50% Aqueous sodium hydroxide                                                                        2.6 L                                                   Darco G-60             900 g                                                  Solka Floc ™       2.5 kg                                                  ______________________________________                                    

Methanesulfonic acid (2.017 kg, 1.36 L, ˜3 equivs.) was added to thestirred solution of the Boc spiroindoline 14 (4.395 kg) in ethanol(total volume ˜25 L) in a reaction vessel at room temperature. The batchwas warmed to 35°-40° C., and stirred overnight. On the next day, thebatch contained ˜1.1 A % of starting material and so the reaction wascontinued for a further 4 hours, then LC showed ratio ofproduct/starting material to be 99.6/0.4. The batch was concentrated invacuo to ˜15 L volume and then diluted with water (44 L). The batch wascooled to 5° C., stirred for 30 minutes and then filtered through aSparkler in-line filter (containing a 10μ cartridge) using a pump toanother vessel to remove a small amount of residual DCU.

The vessel, the pump, the filter and the lines were rinsed with water(10 L), and this was added to the vessel. Ethyl acetate (36 L) was addedto the vessel and the stirred mixture was cooled to 10° C. A solution ofcold (5°-10° C.) 1M aqueous sodium hydroxide solution (16 L) and cold(5°-10° C.) 50% aqueous sodium hydroxide solution (2.6 L) were added at10° C. and the temperature rose to 14° C. The resulting mixture wasstirred for 15 minutes at <14° C. and then the lower aqueous layerseparated off.

The batch was concentrated in vacuo to ˜20 L volume and then a mixtureof ethyl acetate (35 L) and ethanol (5 L) was fed in while maintainingthe volume at ˜20 L. At the end of this distillation the KF was 9160 mgml⁻¹. The batch was solvent switched to ethyl acetate by "feeding andbleeding" ethyl acetate (40 L total). At the end of this distillation,KF was 446 mg ml⁻¹. The batch was diluted with ethyl acetate (10 L).

Darco G-60 (900 g) was added to the hazy mixture. This was rinsed inwith ethyl acetate (6 L). This mixture was stirred at room temperatureovernight. Next day, Solka Floc™ (0.5 kg) was added to the stirred batchin the vessel and then Solka Floc™ (2.0 kg) was stirred in a littleethyl acetate and loaded into an Estrella filter. The excess solvent waspumped away through a Sparkler in-line filter containing a 10μcartridge. The slurry was transferred from the vessel through a filterusing a pump and then through another filter to 2×40 L stainless steelbins. Visual inspection showed the liquors to be clear and clean. Thevessel was rinsed with ethyl acetate (22 L) and this was used to rinsethrough the route outlined above to the stainless steel cans. Thecontents of both cans was transferred into a reaction vessel and thesolution was mixed thoroughly.

The batch (58 L) had a KF of 2950 mg ml⁻¹ and so was redried byconcentrating in vacuo to 20-25 L volume. The batch was diluted to 46 Lvolume (dipstick) by the addition of ethyl acetate (25 L). The KF was363 mg ml⁻¹. The batch was diluted to 62 L volume by the addition ofethyl acetate (17 L) and was used for the final stage of the process.

EXAMPLE 15A ##STR41## Spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamideMethanesulfonate (16)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Amine (15)           2340 g (4.43 moles)                                      Methane sulfonic acid (MsOH)                                                                        316 mL (4.88 moles)                                     EtOAc                 60 L                                                    EtOH                   4.8 L                                                  8% EtOH in EtOAc      20 L                                                    ______________________________________                                    

The volume of the solution of 15 from the previous step was adjusted to60 L with ethyl acetate and EtOH (4.8 L) was added. The MsOH (316 mL)was added in 3 L of EtOAc at 45° C. To the deep red homogeneous solutionwas added 496 g of the title compound Form I seed (10% seed based on theweight of the free amine was employed). The temperature rose to ca. 48°C. and the reaction was aged at 52° C. for 1.5 hours. Analysis indicatedcomplete conversion to the title compound (Form I). (At less than 10%seed longer age (>3 hours) was required). The slurry was allowed to coolto 20° C. overnight and was filtered in a centrifuge under N₂. The cakewas washed with 20 L of 8% EtOH in EtOAc. N₂ is essential duringfiltration because the wet crystals are very hygroscopic. The batch wasdried at 35° C. under vacuum to afford 2.7 Kg (56% overall yield) of thetitle compound (Form I) (99.9 A % purity; <0.1% enantiomer).

The conversion of Form II to Form I is also accomplished where the saltis formed in EtOAc-EtOH by addition of MsOH as above and the initialsolution of the salt (at 55° C.) is cooled to 45° C. Crystals startappearing at that temperature and the slurry becomes thicker with time.The temperature is then raised to 51° C. and the slurry is agedovernight. Complete conversion to Form I of 16 should be expected. Thisprocedure may also be employed to prepare seed crystals of Form I of 16.

EXAMPLE 15B ##STR42## Spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamideMethanesulfonate (16)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Amine (15)              3.1 kg (5.86 mol)                                     Methanesulfonic acid   620 g (6.45 mol)                                       Ethyl acetate           37 L                                                  Absolute ethanol        8.7 L                                                 Spiro 3H-indole-3,4'-piperdin!-1'-yl)-                                                                70 g (0.11 mol)                                       carbonyl!-2-(phenylmethyl-oxy)ethyl!-                                         2-amino-2-methylpropanamide                                                   methanesulfonate (Form I)                                                     ______________________________________                                    

Absolute ethanol (6.4 L) was added to the solution of the amine (15)(3.1 kg) in ethyl acetate (total volume ˜62 L) in a reaction vessel. Thebatch was warmed to 50° C. and a solution of methanesulfonic acid (620g, 412 ml, 1.1 equivs.) in ethyl acetate (11 L) was added over ˜5minutes at 50°-54° C. The batch was seeded with spiro3H-indole-3,4'-piperdin!-1'-yl)-carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamidemethanesulfonate (Form I) (70 g) and the resulting slurry was stirredand heated at 55° C. under nitrogen atmosphere overnight.

The next day, the slurry was cooled to 15°-20° C., held for 2 hours andthen dropped to the 50 cm polypropylene filter under nitrogenatmosphere. The solid product was washed with a mixture of absoluteethanol (2.3 L) in ethyl acetate (26 L). The white, solid product wasdug off and dried in an Apex oven in vacuo at 35° C. for an appropriatetime (approx. two days). The dried spiro3H-indole-3,4'-piperdin!-1'-yl)-carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamidemethanesulfonate (3.352 kg) was sieved using a Jackson-Crockatt sieve togive 3.347 kg (including seed, 70 g)} yield=3.277 kg.

Form I of N- 1(R)- (1,2-dihydro-1-methanesulfonyl-spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamidemethanesulfonate is an anhydrous polymorph characterized by thefollowing properties:

a melting point of 168°-171° C. and solubility in isopropanol of 4.6mg/mL.

The DSC curve for Form I of N- 1(R)-(1,2-dihydro-1-methanesulfonyl-spiro3H-indole-3,4'-piperdin!-1'-yl)carbonyl!-2-(phenylmethyl-oxy)ethyl!-2-amino-2-methylpropanamidemethanesulfonate at 10° C./min in an open cup under nitrogen flowexhibits a single endotherm, due to melting, with a peak temperature ofabout 180° C. and an extrapolated onset temperature (melting point) ofabout 170° C. with an asociated heat of approximately 53 J/g.

Form I was characterized by an X-ray powder diffraction pattern withreflections at approximately: 6.5°, 14.7°, 16.9°, 17.1°, 17.9°, 19.5°,21.1°, 21.7°, and 22.0° (2 theta). Data collected using a PhilipsAPD3720 Automated Powder Diffraction instrument with copper Kαradiation. Measurements were made from 2° to 40° (2 theta) with thesample maintained at ambient room temperature.

HPLC Conditions

LC Retention times on Zorbax RX-C8 (4.6 mm×25 cm), λ=210 nm, flowrate=1.5 ml/min.

Compound 1: 60:40 CH₃ CN-H₂ O (1% H₃ PO₄) RT=5.0 min

Compound 1b: 35:65 CH₃ CN-H₂ O (0.1 w % NH₄ OAc) RT=6.2 min.

Compound 10: 60:40 CH₃ CN-H₂ O (0.1 H₃ PO₄) RT=2.9 min.

Compound 11: 60:40 CH₃ CN-H₂ O (0.1% H₃ PO₄) RT=5.4 min.

Compound 12: 40:60 CH₃ CN-H₂ O pH 5.25 NaH₂ PO₄ (6.9 g/L of H₂ O)(adjust pH with NaOH)!RT=5.6 min

Compound 14: 60:40% CH₃ CN-H₂ O (0.1% H₃ PO₄) RT=4.65 min

Compound 15: 40:60% CH₃ CN-H₂ O pH=5.25 NaH₂ PO₄ (6.9 g/L of H₂ O)!adjust pH with NaOH)RT=4.9 min

LC Retention times on Zorbax RX-C8 (4.6 mm×25 cm), λ=210 nm, flowrate=1.2 ml/min, column temperature=48° C. Solvent A=0.05% Phosphoricacid+0.01% Triethylamine in water Solvent B=Acetonitrile

Gradient system:

    ______________________________________                                        Time           % A    % B                                                     ______________________________________                                         0 min         95     5                                                       35 min         10     90                                                      38 min         95     5                                                       40 min         95     5                                                       ______________________________________                                                      Retention time (mins)                                           ______________________________________                                        Compound 1    25.2                                                            Compound 1b    8.5                                                            Compound 10   20.5                                                            Compound 11   26.3                                                            Compound 12   14.8                                                            Compound 14   25.6                                                            Compound 15   15.7                                                            ______________________________________                                    

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, reaction conditions other than the particular conditions asset forth herein above may be applicable as a consequence of variationsin the reagents or methodology to prepare the compounds from theprocesses of the invention indicated above. Likewise, the specificreactivity of starting materials may vary according to and dependingupon the particular substituents present or the conditions ofmanufacture, and such expected variations or differences in the resultsare contemplated in accordance with the objects and practices of thepresent invention. It is intended, therefore, that the invention bedefined by the scope of the claims which follow and that such claims beinterpreted as broadly as is reasonable.

What is claimed is:
 1. A process for the preparation of a compound offormula V: ##STR43## which comprises: (1) coupling an amino acid of theformula: ##STR44## with a compound of the formula: ##STR45## in thepresence of an acid activating agent which is selected from: DCC and EDCin a solvent which comprises isopropyl acetate and water in the presenceof HOBT to give a compound of formula I: ##STR46## wherein L is an aminoprotecting group, followed by: (2) reacting the compound of the formulaI with a first amino deprotecting agent to give a compound of formulaII: ##STR47## followed by: (3) coupling an amino acid of the formula:##STR48## wherein L is an amino protecting group, with the compound offormula II in the presence of an acid activating agent which is selectedfrom: DCC and EDC in a solvent which comprises isopropyl acetate andwater in the presence of HOBT, to give a compound of the formula III:##STR49## wherein L is an amino protecting group, followed by: (4)reacting the compound of the formula III with a second aminodeprotecting agent to give a compound of the formula IV, or apharmaceutically acceptable salt thereof: ##STR50## followed by:reacting the compound of the formula IV with methanesulfonic acid togive the compound of formula V.
 2. A process for the preparation of acompound of formula I: ##STR51## wherein L is an amino protecting group,by coupling an amino acid of the formula: ##STR52## with a compound ofthe formula: ##STR53## in the presence of an acid activating agent whichis selected from: DCC and EDC in a solvent which comprises isopropylacetate and water in the presence of HOBT, to give the compound offormula I.
 3. The process of claim 2 wherein the acid activating agentis DCC.
 4. The process of claim 2 wherein the solvent additionallycomprises a solvent which is selected from the group consistingof:acetonitrile; ethyl acetate; propionitrile; chlorinated hydrocarbonsselected from dichloromethane, chloroform, carbon tetrachloride,dichloroethane, chlorobenzene, ortho-dichloro-benzene; benzene; toluene;xylenes; and mixtures thereof.
 5. The process of claim 2 wherein thesolvent additionally comprises acetonitrile.
 6. The process of claim 2wherein the temperature of the reaction is between 20° and 35° C.
 7. Theprocess of claim 2 wherein the compound of formula I, the aminoprotecting group is selected from:t-butoxy-carbonyl.
 8. The process ofclaim 2 which is conducted in situ without isolation of the compound offormula I following its preparation.
 9. A process for the preparation ofa compound of the formula II of claim 1: ##STR54## which comprisesreacting a compound of the formula I: ##STR55## wherein L is an aminoprotecting group, with an amino deprotecting agent to give the compoundof formula II.
 10. The process of claim 9 wherein the compound offormula I, the amino protecting group is selectedfrom:t-butoxy-carbonyl.
 11. The process of claim 9, wherein the aminodeprotecting agent is methanesulfonic acid.
 12. The process of claim 9which is conducted in situ without isolation of the compound of formulaII following its preparation.
 13. A process for the preparation of acompound of the formula III: ##STR56## wherein L is an amino protectinggroup, by coupling an amino acid of the formula: ##STR57## wherein L isan amino protecting group, with a compound of the formula II: ##STR58##in the presence of an acid activating agent which is selected from: DCCand EDC in a solvent which comprises isopropyl acetate and water in thepresence of HOBT, to give the compound of formula III.
 14. The processof claim 13 wherein the acid activating agent is DCC.
 15. The process ofclaim 13 wherein the solvent is isopropyl acetate:water.
 16. The processof claim 15 wherein the solvent isopropyl acetate:water is in a ratio ofapproximately 40:60 to 60:40 (by volume).
 17. The process of claim 13wherein the temperature of the reaction is between 20° and 35° C. 18.The process of claim 13 wherein the compound of formula III, the aminoprotecting group is selected from:t-butoxy-carbonyl.
 19. The process ofclaim 13 which is conducted in situ without isolation of the compound offormula III following its preparation.
 20. A process for the preparationof a compound of the formula IV of claim 1, or a pharmaceuticallyacceptable salt thereof: ##STR59## which comprises reacting a compoundof the formula III: ##STR60## wherein L is an amino protecting group,with an amino deprotecting agent to give the compound of formula IV. 21.The process of claim 20 wherein the compound of formula III, the aminoprotecting group is selected from:t-butoxy-carbonyl.
 22. The process ofclaim 20, wherein the amino deprotecting agent is methanesulfonic acid.23. The process of claim 20 which is conducted in a solution comprisingethanol.
 24. The process of claim 20 which is conducted in situ withoutisolation of the compound of formula IV following its preparation.
 25. Aprocess for the preparation of a compound of the formula V: ##STR61##which comprises reacting a compound of the formula IV of claim 1:##STR62## with methanesulfonic acid to give the compound of formula V.26. The process of claim 25 which is conducted in a solution comprisingethyl acetate and ethanol.